Project Summary/Abstract Cells use filopodia, actin-based cellular protrusions, to sense the environment, aid directed migration and serve as the first points of contact for cell-cell interactions. In order for filopodia to initiate, the dendritic actin network needs to be organized into actin bundles perpendicular to the membrane, but how this happens is not well understood. It is known the molecular motor MyTH-FERM (MF) myosin and the actin bundling protein VASP are both required to initiate filopodia. The manner in which MF myosin and VASP are recruited to filopodia initiation sites, and the properties of the molecular motor required for initiation are not known. Dictyostelium is a simple model amoeboid cell which possesses conserved mechanism of filopodia initiation and has a MF myosin (DdMyo7) and a single VASP which are essential for filopodia initiation. The composition of filopodia initiation sites and recruitment dynamics of DdMyo7 and VASP to these sites will be investigated using widefield deconvolution microscopy and PALM. Next, the motor properties including velocity, and processivity of DdMyo7 will be characterized using in vitro motility assays. Next, the effect of motor dimerization and force generation on filopodia initiation will be tested by first characterizing these mutants in vitro, then testing these mutant constructs for rescue of filopodia initiation in vivo. Finally, a stochastic simulation of filopodia initiation has been build and will be expanded to determine which parameters greatly affect initiation. We will use experimental approaches to constrain the simulation, and use simulation to generate more testable predictions. Together, these experiments will bring key insights into the enigmatic molecular details of filopodia initiation, an important process required for directed cell migration.